In 2009, we have advanced the objectives of this project as follows: (1) It is well known that multiple serial passages of a wild-type virulent virus in cell culture often results in the loss of virulence of the virus in a homologous host, which has been a traditional strategy in generating attenuated live viral vaccines including Rotarix vaccine. However, mechanisms underlying this phenomenon are not well understood. The purpose of this study was to monitor phenotypic and molecular characteristics of (i) a mouse-adapted virulent murine rotavirus following serial passages in cell cultures and (ii) a cell culture-adapted avirulent murine rotavirus following serial passages in mouse pups. We passaged a cell culture-adapted avirulent (non-diarrheagenic) murine rotavirus EB strain serially in 4-5 day-old CD-1 mice until the virus became virulent (diarrheagenic). This virulent virus was then inoculated onto primary African green monkey kidney (AGMK) cells and passaged serially 18 times. The 18th cell culture-passaged virus that was shown to be avirulent in mouse pups became virulent again during subsequent serial passage in CD-1 mice. This virulent virus when passaged again serially 18 times in primary AGMK cells became avirulent in mice. Sequencing of all 11 genes of selected virulent and avirulent viruses generated in this study, revealed that virulent viruses when compared with avirulent viruses bore (i) distinct mutations (residues 385, 537, 538 and 558) in gene 4 (encoding outer capsid VP4) and in gene 10 (residues 21 and 37) (encoding viral enterotoxin NSP4) and (ii) no significant mutations in other genes. In addition, no significant difference in virus replication efficiency in mice was observed between virulent and avirulent viruses. We generated 3 baculovirus recombinants expressing the NSP4 protein derived from high (17x) mouse-passaged virulent EB virus (sample A), low (8x) mouse-passaged virulent EB virus (sample B), or cell culture-passaged avirulent EB virus (sample C). Following oral inoculation of various recombinant NSP4 proteins in mouse pups, diarrhea developed in 91.6% (11/12) in sample A group;12.5% (1 of 8) in sample B group;and 0% (0 of 12) in sample C group, indicating that the NSP4 protein played an important role in pathogenesis in this model. (2) To study which of the four mutations (residues 385, 537, 538 and 558) detected in gene 4 between virulent and avirulant murine rotaviruses are associated or not associated with virulence/avirulence of murine rotavirus in mouse pups, we passaged a human diarrheal stool suspension containing G1P8 virus (three strains), G8P4 virus (one strain) or G9P8 virus (one strain) serially up to 40 times in two different cell cultures (primary AGMK cells and human colon adenocarcinoma cell line HT29). Whole genome sequence analysis of six virulent virus strains in stool samples as well as 12 cell culture-passaged virus strains (6 in primary AGMK cells and 6 in HT29 cells) is in progress.